A wide variety of diagnostic techniques are presently available for detection of organisms within a biological sample, including for example, biochemical tests, immunological tests and cytological tests. The majority of these techniques, however, have drawbacks related to length of time, quantity of sample required, labor, training in the use of equipment, expertise level and lack of specificity or sensitivity of detection. Often the biological samples of interest may be limited in terms of the number of cells or quantity of target nucleic acid to be detected, which in turn will affect the sensitivity of the method used. Thus, for successful detection of an organism, it may be necessary to increase or amplify the quantity of target nucleic acids in order to overcome the sensitivity limitation of a small number of target organisms.
One of the most widely used in vitro methods for amplifying selected nucleic acid sequences is the Polymerase Chain Reaction ("PCR", see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202). Briefly, two oligonucleotide primers which flank the DNA segment of the target sequence to be amplified are used to initiate exponential copying of the target sequence. After heat denaturation of the target, hybridization of the primers occurs to their complementary sequences on the opposite strands and replication occurs enzymatically due to elongation of the two primers. Repetitive cycles of denaturation, primer annealing, and extension are carried out, resulting in replication of a complementary strand to each of the original strands per cycle. In turn, each of the product strands is capable of being hybridized to the primers. This results in an exponential amplification of the target nucleic acid which can subsequently be detected.
There are, however, a number of technical problems associated with PCR. For example, false positive results can occur from contaminating nucleic acids arising from a number of sources (Kwok and Higuchi, Nature 339:237-238, 1989; Kitchin et al., Nature 344:201). PCR products from previous amplification of the target can also accumulate in the laboratory, resulting in cross-contamination between different samples. Problems can also arise from the co-amplification of non-specific target caused by hybridization of primers to extraneous sequences along the target template or other heterologous nucleic acids present in the sample. The problem of false negatives is discussed by Niederhauser et al. PCR Methods Appl. 4: 117-123, 1994. The technical ability of laboratory personnel, laboratory capabilities and logistics also have to be taken into consideration.
Problems with heterologous nucleic acid contamination, which may cause inhibition, or cross-over contamination, which gives false positive, affects other amplification technologies such as Nucleic Acid Sequence Based Amplification (NASBA), Gap Ligase Chain Reaction (Gap-LCR), Strand Displacement Amplification (SDA), and Q-Beta Replicase (See generally Carrino and Lee, J. Microbiol. Meth. 23:3-20, 1995; Altwegg, J. Microbiol. Meth. 23:21-30).
A number of these problems can be resolved if, in an amplification system, the target is not amplified. One such method is the cycling probe technology ("CPT", see, e.g., U.S. Pat. Nos. 5,011,769 and 5,403,711), where a specific probe containing a scissile linkage oligonucleotide complementary to the target sequence is utilized.
The present invention discloses novel compositions and methods for use in cycling probe reactions, which are simple, rapid and inexpensive to use. In particular, unlike other nucleic acid amplification technologies, the methods provided herein may be accomplished at a constant temperature, do not require more than one enzyme or probe and can be carried out in the presence of heterologous DNA that may be present in the sample. Further, the present invention provides other related advantages.